INTERFERON-GAMMA MODULATES INTESTINAL P-GLYCOPROTEIN: MOLECULAR MECHANISM(S) AND CLINICAL IMPLICATIONS
Intestinal P-glycoprotein (P-gp) expression and luminal nitric oxide (NO) levels are significantly greater in patients with refractory inflammatory bowel disease (IBD). We investigated whether the proinfammatory cytokine IFN-?, the transcription factor NF-?B, and the signaling intermediate NO regulate the P-gp encoding ABCB1 gene using the Caco-2 cells as an in vitro model of human intestinal epithelial cells. To identify cytokine-mediated activation of signal transduction pathways, phosphorylation of JAK-2, cytosolic I?B? degradation and nuclear binding of NF-?B were determined in Caco-2 cells stimulated with 10 ng/ml of IFN-? using immunoblot analysis and electrophoretic mobility shift assays, respectively. Changes in ABCB1 mRNA were measured by realtime PCR, total cellular P-gp protein was quantitatively assessed using immunoblot analysis, and digoxin uptake was used to determine P-gp efflux activity at the apical membrane. To further evaluate underlying molecular pathways, similar experiments were performed using Caco-2 cells expressing phosphorylation-deficient I?B? (Caco-2/ NF-?B-/-) in the presence or absence of the NF-?B inhibitor parthenolide (25 ìM), the iNOS inhibitor L-NIL (1 mM), and the NO donor SNAP (0.1-5 mM). Transactivation of the ABCB1 promoter was determined using a 1 kb ABCB1-luciferase construct that contained a functional or mutated NF-?B response element. Intracellular NO levels were determined by the Griess reaction. IFN-? stimulation of Caco-2 cells enhanced phosphorylation of JAK-2, cytosolic degradation of I?B?, increased nuclear binding of NF-?B, and augmented NO production by 9-fold. In parallel, ABCB1 promoter activity was increased by 4-fold, ABCB1 mRNA by at least 2-fold, total cellular P-gp protein expression by 2-fold, and digoxin uptake reduced by ~40%. Significant NF-?B activation, NO generation, and changes in ABCB1 transcription and P-gp function were absent in Caco-2/NF-?B-/- or parental Caco-2 cells coincubated with parthenolide and LNIL, respectively. Incubation of Caco-2 cells with SNAP increased NO levels, I?B? degradation, nuclear binding of NF-?B, ABCB1 mRNA levels, and P-gp protein levels by 2-fold while digoxin uptake was reduced by 45%. We conclude that NO and NF-?B are crucial biochemical mediators in IFN-?-induced transcriptional regulation of the ABCB1 gene in this in vitro cell culture model of human intestinal epithelial cells.
School:University of Cincinnati
School Location:USA - Ohio
Source Type:Master's Thesis
Keywords:p glycoprotein nitric oxide nf kappa b inflammatory bowel disease parthenolide abcb1 inducible synthase
Date of Publication:01/01/2005